The regulation of the gene coding for preproenkephalin (ppEnk), the precursor of the opioid peptides methionine- and leucine-enkephalin, is under investigation. A full-length rat ppEnk cDNA clone, constructed and characterized by us last year, was used as a sensitive hybridization probe for ppEnk mRNA in RNA preparations for clonal cell lines of neuronal origin. NG108-15 mouse neuroblastoma x rat glioma hybrid cells contain small amounts of rat ppEnk mRNA, 20-120 fg/ug RNA by Northern blot analysis. Treatment of NG108-15 cells with glucocorticoids such as dexamethasone elevated the ppEnk mRNA abundance to 3 and 9 times the control at 24 h and 8 days, respectively. Treatment of the cells with 8-Br-cAMP or activators of adenyate cyclase such as forskolin had little or no effect on the ppEnk mRNA abundance. However, treatment with both glucocorticoid and either 8-Br-cAMP or forskolin markedly elevated the ppEnk mRNA abundance to 5-8 times and 30 times the control at 24 h and 8 days, respectively. These increases were blocked by actinomycin D, an inhibitor of transcription. The Met-enkephalin content of the cells was increased in parallel with the mRNA abundance. These results demonstrate that the transcription of the rat ppEnk gene is positively regulated by the synergistic interaction of glucocorticoids and cAMP. C6 and C6-BU1 rat glioma cells contain a much higher abundance of ppEnk mRNA (3-6 pg/Mug RNA) but lower enkephalin content than NG108-15 cells. C6 ppEnk mRNA is markedly but transiently elevated by the combination of glucocorticoids and compounds that elevate cAMP, e.g. beta-adrenergic receptor agonists such as norepinephrine and isoproterenol, which activate C6 adenylate cyclase. Glucocorticoids alone were ineffective, while cAMP elevation alone had a slight effect. These results suggest that the ppEnk gene may be expressed in glial cells or their developmental precursors. In collaboration with Dr. H-Y Yang, efforts have begun to clone cDNA for precursor(s) to two anti-analgesic neuropeptides isolated by her.